title: Dissecting partner recognition by an intrinsically disordered protein using descriptive random mutagenesis.
abstract: In view of getting insights into the molecular determinants of the binding efficiency of intrinsically disordered proteins (IDPs), we used random mutagenesis. As a proof of concept, we chose the interaction between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein and assessed how amino acid substitutions introduced at random within NTAIL affect partner recognition. In contrast with directed evolution approaches, we did not apply any selection and used the gene library approach not for production purposes but for achieving a better understanding of the NTAIL/XD interaction. For that reason, and to differentiate our approach from similar approaches that make use of systematic (i.e., targeted) mutagenesis, we propose to call it "descriptive random mutagenesis" (DRM). NTAIL variants generated by error-prone PCR were picked at random in the absence of selection pressure and were characterized in terms of sequence and binding abilities toward XD. DRM not only identified determinants of NTAIL/XD interaction that were in good agreement with previous work but also provided new insights. In particular, we discovered that the primary interaction site is poorly evolvable in terms of binding abilities toward XD. We also identified a critical NTAIL residue whose role in stabilizing the NTAIL/XD complex had previously escaped detection, and we identified NTAIL regulatory sites that dampen the interaction while being located outside the primary interaction site. Results show that DRM is a valuable approach to study binding abilities of IDPs.
authors.affiliation: CNRS and Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, 13288 Marseille, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Gruet
authors.forename: Marion
authors.initials: M
authors.lastname: Dosnon
authors.forename: Andrea
authors.initials: A
authors.lastname: Vassena
authors.forename: Vincent
authors.initials: V
authors.lastname: Lombard
authors.forename: Denis
authors.initials: D
authors.lastname: Gerlier
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology*; D009154:Mutation / Q000502:physiology; D019151:Peptide Library; D011485:Protein Binding; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: DRM; GFP; IDP; Intrinsically disordered protein; MeV; MoRE; descriptive random mutagenesis; green fluorescent protein; library; measles virus; molecular recognition element; partner recognition; protein complementation assay; protein–protein interactions; split-GFP reassembly